Biosensors include enzyme sensors, immunosensors, and bacteriological sensors, and are considered to be very important for measuring material properties or substances (hereinafter referred to as targets) in a wide variety of fields such as the fields of medicine, food, and industry.
For instance, a glucose sensor measures the blood sugar level from the concentration of glucose in the blood. The glucose sensor has an electrode and an enzyme membrane which covers the electrode. Glucose oxidase (GOD) is fixed on the enzyme membrane as a receptor which specifically reacts with glucose.
Glucose is oxidized by GOD in the glucose sensor according to the reaction formula (1), and is broken down into gluconic acid and hydrogen peroxide.
(Formula 1)C6H12O6+O2→C6H10O6+H2O2  (1)
Next, the hydrogen peroxide produced in the reaction formula (1) is diffused in the solution between the electrode and the enzyme membrane to the electrode surface, and undergoes electrolysis at the electrode as shown in reaction formula (2), and the electrons are transferred to the electrode.
(Formula 2)H2O2→*2H++O2+2e−  (2)
A proportional relationship exists between the glucose concentration, the diffusion of glucose to the enzyme membrane, and the diffusion of hydrogen peroxide in the solution, and the glucose concentration can be obtained by measuring the electrical current from the electrolysis reaction of reaction formula (2). However, with this type of glucose sensor, the transfer of electrons to the electrode from the enzyme membrane is dependent on the diffusion speed and the diffusion concentration or the like of the hydrogen peroxide to the electrode, so rapid measurements and measurements with high electrical currents are difficult.
Therefore, biosensors are being developed which transfer a charge between a receptor and the electrode using a mediator. FIG. 7 shows the operating principle of a mediator type biosensor, and the following reaction takes place between the mediator M, an enzyme E, and a substrate S which reacts with the enzyme E.
Electrons are transferred by the oxidation reduction reaction between the oxidized enzyme Eox and the substrate S, and reduced enzyme Ered and product P are produced. Next, oxidized enzyme Eox and reduced mediator Mred are produced by the oxidation reduction reaction between the reduced enzyme Ered and the oxidized mediator Mox. Finally, oxidized mediator Mox is produced and electrons are transferred to the electrode by the oxidation reduction reaction between reduced mediator Mred and the electrode. In other words, the electrons generated by the enzyme reaction are rapidly transferred in high-volume from the enzyme to the electrode through the mediator. At this time, oxidation and reduction is repeatedly occurring between the enzyme E and the mediator M. The aforementioned enzyme reaction was for the case where the substrate S is oxidized and electrons are transferred to the electrode, but if the substrate S is reduced and electrons are consumed, the electrons will transfer to the enzyme from the electrode by the reverse cycle.
FIG. 8 shows the structure of a mediator type biosensor with an electrode which uses C60 fullerene as shown in the Journal of Electroanalytical Chemistry 454, 9-13, 1998. The C60 fullerene 2, which is the mediator, is fixed to the electrode 1 by a self organizing monomolecular film 3 made from -HS—(CH2)2—NH2. The surface of the C60 fullerene 2 is modified with ═CH—COOH in order for the C60 fullerene 2 and the self organizing monomolecular film 3 to bond. Furthermore, the enzyme 4 and the C60 fullerene are not bonded, and the enzyme 4 is suspended in the solution.
With the biosensor described in the aforementioned documentation, electrons are generated by the oxidation reduction reaction between the glucose, which is the substrate 5, and the GOD, which is the enzyme 4, and the electrons are transferred to the electrode 1 through the C60 fullerene 2, and the glucose concentration is measured.
The mediator type biosensor shown in the aforementioned documentation uses C60 fullerene 2, which has excellent properties for electron attracting and electron donating. However, the enzyme 4 is suspended in the solution and is not bonded with the C60 fullerene 2. Therefore, even when electrons are transferred by the oxidation reduction reaction between the enzyme 4 and the substrate 5, if the enzyme 4 and the C60 fullerene 2 are not bonded, the C60 fullerene 2 cannot transfer a large volume of electrons at high-speed from the enzyme 4 to the electrode 1.
Furthermore, modifying groups are required on the surface of the C60 fullerene 2 in order to fix the C60 fullerene 2 to the electrode 1. Therefore, the electron distribution of the Π bonds, which contribute to the transmission of electricity, will be inconsistent, and there will be problems with a loss of the properties of C60 fullerene 2, including electron attracting and electron donating.
Therefore, an object of the present invention is to provide a target recognizing element wherein a receptor is fixed to an inclusion complex which includes a mediator.
Furthermore, another object of the present invention is to provide a biosensor wherein the position of the mediator is fixed with regard to the electrode without using modifying groups on the surface of the mediator.